Glutaminase (L-Glutamine amidohydrolase, EC3.5.1.2, hereinafter, referred to as glutaminase) is an enzyme that hydrolyzes glutamine into glutamic acid and ammonia. Glutaminase is known to play an important role in the food-processing industry, particularly when food flavorings, such as soy sauce, which is obtained by enzymatic degradation of protein, are produced. When soy sauce is produced, a protein, as a raw material, is degraded into peptides and finally into constitutive amino acids by the action of various proteinases produced by Aspergillus. Glutamic acid, which is a kind of constitutive amino acid, plays a central role among flavor-components of soy sauce. Two pathways have been devised to generate glutamic acid in soy sauce manufacture. One pathway is the direct generation of glutamic acid in the above described degradation process of the protein raw material (1st pathway) and the second pathway is the generation of glutamic acid by converting the glutamine produced in the degradation process of the protein raw material into glutamic acid by the action of glutaminase (2nd pathway). Protein-storing plants including soya beans that are the raw materials in soy sauce manufacture are rich in acidic amino acid, such as glutamic acid and asparatic acid. Most acidic amino acids are known to exist in the form of amide, such as glutamine and asparagine. In addition, glutamine generated by the degradation of raw materials in the soy sauce brewing process changes non-enzymatically and relatively quickly into tasteless pyroglutamic acid. Therefore, in soy sauce brewing, primary importance has been placed on the reaction which converts glutamine into glutamic acid (the 2nd pathway).
In soy sauce manufacture, the use of yellow koji molds (e.g. Aspergillus sojae) capable of high production of glutaminase has been shown to cause increases in the amount of glutamic acid in soy sauce moromi, which is formed after the raw material for soy sauce production is treated before fermentation (see Yamamoto et. al. J. Ferment. Technol., Vol. 52, No. 8, 564–569 (1974)), and that within this soy sauce moromi, there exists a correlation between the glutaminase in the insoluble fractions (also described as fractions of cell surface and cell first-surface, intracellular fractions, fractions of microbial body surface and fractions within the microbial body) of yellow koji molds and glutamic acid (see Nippon Shoyu Kenkyusho Zasshi Vol. 5, No. 1, 21–25, 1979). Thus, the glutaminase produced by koji molds has been recognized as important.
Aspergilli (including yellow koji molds) Aspergillus oryzae and Aspergillus sojae, have traditionally been used in the production of brewed food in Japan, such as miso, soy sauce and old-fashioned sake. These microbes are particularly important industrially, because of their high productivity of enzymes and high reliability of safety as attested by their long-standing use.
Some of the glutaminases produced by the microbes of the genus Aspergillus including these yellow koji molds have been purified and their properties reported. Intra- and extra-microbial glutaminases have also been purified from Aspergillus oryzae and their properties studied (see Yano, T et al, J. Ferment. Technol., Vol. 66, No. 2, 137–143 (1988)). All of these glutaminases have molecular weights of approximately 113,000 and they all have similar properties. Further, 2 types of extra-microbial glutaminases, which are different from the above types, have been purified from Aspergillus oryzae, and their properties have been studied (see WO99/60104, and JP Patent Publication (Unexamined Application) No. 2002-218986). The genes of these extra-microbial glutaminases have been isolated and analyzed. Further, extra-microbial glutaminases have been purified from Aspergillus sojae and their genes have been reported (see JP Patent Publication (Unexamined Application) No. 2000-166547). Furthermore, a glutaminase gene which is different from the above genes has also been isolated from Aspergillus sojae (see Japanese Patent Application No. 2001-187433).
The localization of the glutaminase of yellow koji molds has been studied. Fractions from 3 broadly classified locations: extracellular, cell surface and intracellular (cell membrane and cytoplasm), have been studied for their properties and distribution ratios using crude enzyme solutions. As a result, it has been reported that most glutaminases are located on the cell surface and intracellularly (see Nippon Shoyu Kenkyusho Zasshi Vol. 11, No. 3, 109–114, 1985).
Glutaminases derived from yellow koji molds and glutaminase genes that have been reported so far are all extra-microbial glutaminases. Based on analysis of molecular weight and enzymatic properties, however, it has been suggested that the only intra-microbial glutaminase, as the one obtained by Yano et al., may be identical to extra-microbial glutaminase. No glutaminase and genes thereof shown to be located definitely on the cell surface and intracellularly have been reported. As described above, insoluble glutaminase is known to have an effect in soy sauce manufacture, so that isolation of these glutaminases is strongly desired. Further, the use of these glutaminases has been limited in terms of industrial use, because of their low quantity of production and the like. Hence, isolation of the gene that enables mass-preparation of such glutaminases is also strongly desired.
Among the production methods for food flavorings involving enzymatic degradation of proteins, an extremely effective and efficient method for efficiently producing food flavorings having rich glutamic acid content involves the degradation of proteins at high temperature, because high temperature enhances the degradation rate of protein and prevents contamination by saprophytes. However, as the temperature rises, the efficiency of glutamine to pyroglutamic acid conversion also increases, glutaminase thus must act quickly. Therefore, high optimal temperature and thermostability are required for glutaminase to be utilizable under such conditions. Hence, isolation of glutaminase having these properties has been strongly desired.